Johan Kamphuis

The First Water-Soluble 310-Helical Peptides

Two water-soluble 3(10)-helical peptides are synthesized and fully characterized for the first time. The sequence of these terminally blocked heptamers comprises two residues of the Calpha-trisubstituted alpha-amino acid 2-amino-3-[1-(1,4,7-triazacyclononyl)]propanoic acid and five residues of a Calpha-tetrasubstituted alpha-amino acid (either alpha-aminoisobutyric acid or isovaline). Using CD and NMR techniques we were able to show that both heptapeptides are well structured in water, and that the type of conformation adopted is indeed the ternary 3(10)-helix.

Enzymatic resolution of 1‐phenyl‐1,2‐ethanediol by enantioselective oxidation: Overcoming product inhibition by continuous extraction

Oxidations of alcohols by alcohol dehydrogenases often suffer from low conversions and slow reaction rates due to severe product inhibition. This can be overcome by continuous product extraction, because only the concentrations, but not the kinetic parameters, can be changed. As a consequence, it is favorable to apply a differential circulation reactor with continuous product extraction, where only a small amount of product is formed per cycle. The product is then directly extracted using a microporous hydrophobic hollow fiber membrane. This results in an increase of the relative activity of the dehydrogenase at a given conversion. The reaction investigated is the kinetic resolution of racemic 1‐phenyl‐1,2‐ethanediol by glycerol dehydrogenase (GDH). The resulting oxidation product, 2‐hydroxyacetophenone, causes a strong product inhibition. Additionally, it reacts in a chemical reaction with the cofactor lowering its active concentration. Because the GDH needs β‐nicotinamide adenine dinucleotide (NAD+) as a cofactor, lactate dehydrogenase is used to regenerate NAD+ from NADH by reducing pyruvate to (L)‐lactate. A conversion of 50% with respect to the racemate and an enantiomeric excess >99% of the (S)‐enantiomer was reached.

Enzymatic resolution of α,α-disubstituted α-amino acid esters and amides

The scope and limitations of the enzymatic resolution of α,α-disubstituted α-amino acid amides by an amino acid amidase from Mycobacterium neoaurum and of the corresponding ethyl esteis with Pig liver estetase (PLE) have been studied. Moderate enantiomeric excesses were obtained with PLE, with only a narrow substrate specificity. Mycobacterium neoaurum on the contrary yields a broad range of S-α,α-disubstituted α-amino acids 1 and the corresponding R-amides 2.

Nitroxyl peptides as catalysts of enantioselective oxidations.

The achiral, nitroxyl-containing alpha-amino acid TOAC (TOAC = 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), in combination with the chiral alpha-amino acid C(alpha)-methyl valine [(alphaMe)Val], was used to prepare short peptides (from di- to hexa-) that induced the enantioselective oxidation of racemic 1-phenylethanol to acetophenone. The best catalyst was an N(alpha)-acylated dipeptide alkylamide with the -TOAC-(alphaMe)Val- sequence folded in a stable, intramolecularly hydrogen-bonded beta-turn conformation with large, lipophilic (hydrophobic) N- and C-terminal blocking groups. We rationalized our findings by proposing models for the diastereomeric intermediates between (R)-[and (S)]-1-phenylethanol and the catalyst Fmoc-TOAC-L-(alphaMe)Val-NHiPr, based on the X-ray diffraction structure of the latter.

Ochrobactrum anthropi NCIMB 40321 : a new biocatalyst with broad-spectrum L-specific amidase activity

Of 125 microorganisms that were able to use α-hydroxy acid amides as sole nitrogen source, Ochrobactrum anthropi NCIMB 40321 was selected for its ability to hydrolyse racemic amides l-selectively. The substrate specificity of whole O. anthropi cells is remarkably wide and ranges from α-H-α-amino-, α-alkyl-α-amino, N-hydroxy-α-amino acid amides to α-hydroxy-acid amides. After 50% conversion, both the l-acids formed and the remaining d-amides were present in >99% enantiomeric excess, and ammonia accumulated in stoichiometric amounts. Using mandelic acid amide as a model substrate, the hydrolysis was optimized. Optimal rates were observed at pH 8.5 at 50°C. At higher temperatures the initial rate was even higher; however, fairly rapid inactivation occurred.

Preferred conformation of peptides rich in alicyclic Cα,α-disubstituted glycines

The conformational preferences of the alicyclic Cα,α‐disubstituted glycines Acnc (1‐amino‐1‐cycloalkane‐carboxylic acid; n = 4, 7, 9, 12) were assessed in selected model compounds, including homopeptides and Ala (or Aib, α‐aminoisobutyric acid)/Acnc peptides containing a small total number of residues, by Fourier transform ir absorption, 1H‐nmr, and x‐ray diffraction analyses. The results obtained indicate that β‐turn and 310‐helical structures are preferentially adopted by short peptides rich in these cycloaliphatic α‐amino acids.

Production of 1-kestose with intact mycelium of Aspergillus phoenicis containing sucrose-1F-fructosyltransferase

SummaryFavourable reaction conditions for the enzymatic production of 1-kestose by sucrose-1F-fructosyltransferase, SFT (EC 2.4.1.99) from Aspergillus phoenicis CBS 294.80 mycelium were established. The intracellular enzyme SFT works best at 60°C, exhibits a relatively high thermostability and possesses an alkaline pH optimum. An invertase also present in the mycelium of A. phoenicis possesses an acidic pH optimum. Consequently, around pH 8.0 sucrose is converted mainly to 1-kestose and nystose while fructose is only formed in relatively small amounts. Under optimal conditions (55° C, pH 8.0 and an initial sucrose concentration of 750 g 1-1) a yield of about 300 g 1-kestose per 1.01 reaction mixture could be achieved after 8 h.

The potential of lyases for the industrial production of optically active compounds

Lyases catalyse the cleavage of C-C, C-N, C-O and other bonds by elimination to produce double bonds or, conversely, catalyse the addition of groups to double bonds. These enzymes do not require cofactor recycling, show an absolute stereospecificity and can give a theoretical yield of 100%, compared with only 50% for enantiomeric resolutions. Lyases are therefore attracting considerable interest as biocatalysts for the production of optically active compounds, and have already found application in several large commercial processes.

Aspartame dipeptide analogues: effect of number of side-chain methylene group spacers and Cα-methylation in the second position

Abstract Our model of the active site of the sweet taste receptor is shown to be consistent with the aspartame analogues in which the L-Phe 2 residue is replaced by L-(αMe)Phg, L-(αMe)Phe or L-(αMe)Hph. The analogues containing either the first or the third C α -methylated, phenyl-containing residue in the second position of the dipeptide were synthesized and found to be approximately as sweet as aspartame itselfand its L-(αMe)Phe 2 analogue.

The p-bromobenzamido chromophore as a circular dichroic probe for the assignment of the screw sense of helical peptides

Abstract The para -bromobenzoyl group linked to the N-terminus of a helical peptide chain turned out to be useful as a CD probe in the determination of the relationship between C α -configuration of coded and non-coded α-amino acids and peptide helix screw sense in solution. The results of the present conformational investigation agree well with published crystal-state structural data on the same peptides obtained using X-ray diffraction where the para -bromobenzoyl group helped us to solve the phase problem by virtue of its heavy atom (bromine).